Bacterial PhyA protein-tyrosine phosphatase-like myo-inositol phosphatases in complex with the Ins(1,3,4,5)P4 and Ins(1,4,5)P3 second messengers

Abstract

myo-Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃ Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo-inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP₆ or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P₅, have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P₅ These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended Ω-loop, and a β-hairpin turn of the Phy-specific domain.

Keywords: PTP-like; X-ray crystallography; complex; cysteine phosphatase; inositol 1,4,5-trisphosphate (IP3); inositol phosphate; phosphatase; protein complex; second messenger; substrate specificity.

Figure 1.

Ribbon diagrams of the overall fold of PhyAsrC252S (gray) and PhyAmmC250S/C548S (brown/blue) in complex with Ins(1,3,4,5)P₄. A, PhyAsrC252 is oriented so that the Ins(1,3,4,5)P₄ ring and phosphoryl groups are clearly visible, with the C1-phosphoryl group bound to the phosphate-binding loop (P-loop, yellow). B, the individual N-terminal (brown) and C-terminal (blue) repeats of PhyAmm are shown in the same orientation as in A. C, PhyAmm oriented to view the tandem repeats and linker resulting in the Ins(1,3,4,5)P₄ ligand viewed from the top with the P-loop below the ligand. The ligand atoms are shown as sticks with carbon shown in gray, oxygen in red, and phosphorus in orange. The variable loops are shown in green. These include the extended loops prior to the penultimate helix, the α-turn (N-terminal repeat) and β-hairpin turns (PhyAsr and C-terminal repeat), and the extended Ω-loops. The P-loop (yellow) and the general acid loop (GA-loop, cyan) contribute catalytic residues. Mutation of the cysteine nucleophile to the isosteric serine prevents thiolate formation and renders the enzyme inactive.

Figure 2.

Clear electron density for the phosphoryl groups and C2-hydroxyl allows for an unambiguous fit of the ligands and places the C1-phosphoryl group (P1) above the cysteine to serine mutations at positions 252 (PhyAsr) and 548 (PhyAmm). The refined 2mF_o − DF_c electron density is contoured at 1 σ (blue mesh) for PhyAsrC252S in complex with Ins(1,3,4,5)P₄ (A) and Ins(1,4,5)P₃ (B) and for the PhyAmmC252S/C548S C-terminal repeat in complex with Ins(1,3,4,5)P₄ (C). Ligand and protein are shown as sticks, with oxygen shown in red, nitrogen in blue, phosphorus in orange, and carbon in gray.

Figure 3.

PhyAsrC252S and the C-terminal repeat of PhyAmmC250S/C548S bind the IP substrates nearly identically using a subset of the contacts identified in the PhyAsrC252·InsP₆ (PDB 3MMJ) structure (17). The phosphoryl-binding sites are labeled according to the InsP₆ structure (P_s, P_b, P_a′, and P_c). A, stereo view of the superposition of PhyAsrC252S (gray) and the C-terminal repeat of PhyAmmC250S/C548S (blue) in complex with Ins(1,3,4,5)P₄. B, PhyAsrC252S in complex with Ins(1,4,5)P₃ in the same conformation as in A less one phosphoryl group. C, the N-terminal active site does not bind Ins(1,3,4,5)P₄ in a catalytically competent manner. Instead, the ligand is more than 8 Å (long dashed line) from the inorganic phosphate bound by the P-loop. The short dashed lines represent the distances between the phosphoryl group bound by the P-loop and the serine hydroxyl. Residues that interact with the ligands are derived from the P-loop (yellow), GA-loop (cyan), Phy domain, and penultimate helix. Oxygen is shown in red, nitrogen in blue, phosphorus in orange, and carbon in gray.

Figure 4.

The superposition of PhyAsrC252S in complex with InsP₆ (blue) (PDB 3MMJ) and Ins(1,3,4,5)P₄ (orange) demonstrates the 180° rotation and tilt of the myo-inositol ring toward the GA-loop of the less-phosphorylated IPs relative to InsP₆. The P-loop, GA-loop, and Tyr-309 of the protein are shown. The myo-inositol ring and only two of the phosphoryl group are displayed to simplify the diagram. Despite the rotation and tilt of the myo-inositol ring, the C1-phosphoryl group of Ins(1,3,4,5)P₄ remains bound by the P-loop, and the remaining phosphoryl groups are bound by equivalent residues. For example, the C5-phosphoryl groups in these structures form similar hydrogen bonds with Tyr-309 that originates from the opposite side of the residue.

Figure 5.

Variable loops implicated in the substrate specificity of PTPLPs. A, stereo view of the superposition of PhyAsrC252S^.Ins(1,3,4,5)P₄ (blue), PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ (red), and PhyAbb (green, PDB 4NX8) as a ribbon diagram with the ligand as sticks. The variable loops (colored segments) that influence substrate specificity include the extended loop prior to the penultimate helix, the extended Ω-loop, and the β-hairpin turn within the Phy-specific domain. B, the PhyAsr (blue) active site is relatively occluded (RO) on the P_a/P_b side and relatively accessible (RA) on the P_a′/P_b′ side. C, in the case of PhyAmm (red), the relatively occluded and relatively accessible sides are reversed. D, PhyAbb (green) has a 13-residue deletion, which removes the loop that contains the β-hairpin turn, resulting in an accessible (RA) active site on the P_a′/P_b′ side. Additionally, the position of the extended loop prior to the penultimate helix of PhyAbb is similar in position to the equivalent loop of PhyAmm, and the extended Ω-loop is deleted, leaving the P_a/P_b side more accessible (RA). As a result, PhyAbb produces four different InsP₄ products in contrast to PhyAsr and PhyAmm.

Figure 6.

HPIC chromatograms of the PhyAsr (A), PhyAmm (B), and PhyAbb InsP₆ (C) hydrolysis products demonstrating that the hydrolysis pathways diverge to produce alternate InsP₄ products. InsP₆ (5 mm) was incubated with 10 nm PhyAsr and PhyAmm at room temperature and 100 nm PhyAbb with 10 mm InsP₆. Samples were taken at 0 min (blue), 20 min (orange), 30 min (red), 40 min (green), 50 min (purple), and 60 min (cyan) and separated using a CarboPac PA-100 analytical column with a methanesulfonic acid gradient (37). IPs were visualized using a post-column reactor with 0.1% (m/v) Fe(NO₃)₃ in a 2% (m/v) HClO₄ solution (0.2 ml/min).